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At least 703 records · Page 39Linked to original sources

PubMed1993

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Deletion↗

PubMed1975

An electrophoretic study of the distribution and properties of human hexokinases.

An electrophoretic system which gives a clear separation of human hexokinases HK1, HK2 and HK3 is described. The distribution of the hexokinase isozymes in various human tissues, both adult and fetal, is reported. Some properties of the isozymes were investigated. HK2 was found to be more thermolabile than HK1, and there was also a small but significant difference in molecular size. Unlike HK3, HK1 and HK2 are not inhibited by high glucose concentrations. Screening of red cell lysates from 800 unrelated European individuals revealed no genetic variants of HK1 and HK2. However, in view of their difference in properties, it seems probable that the HK1 and HK2 isozymes are determined by separate gene loci.

Brain↗

PubMed1987

Genetic studies on hexokinase in the mosquito Aedes togoi.

Hexokinases (EC 2.7.1.1) were genetically analyzed in the mosquito Aedes togoi by agar gel electrophoresis. Enzyme activity was observed anodally in one major banding region (HK-1) on the gel and in another faintly stained region (HK-2). A total of six bands was detected in the HK-1 region. All six bands could be detected in three body parts, head, thorax, and abdomen, of adults with different banding intensities. The third and fourth bands, numbered from the more anodal side, showed the broadest substrate specificity and the greatest enzyme activity throughout development. Genetic analysis of the six HK-1 bands was undertaken on the hypothesis of a single gene locus (or three extremely tightly linked loci). The analysis gave the following gene order: HK-1--4.2 +/- 1.8 (recombination units +/- SE)--To-2--Odh-2--29.5 +/- 2.5--sex (M/m)--s. A comparison is made of gene loci for hexokinases among the mosquito species Culex pipiens, Aedes aegypti, and this species, along with a comment on linkage relationships between Hk and Odh (octanol dehydrogenase) loci in three Aedes species.

Aedes↗

PubMed1984

The ubiquitous localization of type I hexokinase in rat peripheral nerves, smooth muscle cells and epithelial cells.

An indirect immunoperoxidase technique has been used to determine the localization of type I hexokinase in a wide variety of Carnoy-fixed, paraffin-embedded rat tissues. The results suggest that the widespread tissue distribution of the isoenzyme is due to its ubiquitous localization in the nervous, smooth muscle and epithelial components of each tissue. The majority of the immunostaining was confined to cells with substantial energy requirements which are probably mainly satisfied through the breakdown of glucose. This observation is consistent with the known predominance of type I hexokinase in the central nervous system and with the regulatory role allotted to it in this tissue.

Animals↗

PubMed1984

Histochemical and immunohistochemical localization of hexokinase isoenzymes in rat kidney.

Histochemical and immunohistochemical staining techniques have been used to investigate the localization of hexokinase isoenzymes within rat kidney tissue. Hexokinase type I was shown to be the major isoenzyme present. It was located mainly in the thin and thick limbs of loops of Henle, in distal tubules and in the transitional or dark cells in the initial portions of collecting ducts. The smooth muscle cells of arteries and arterioles, peripheral nerves and the transitional epithelial cells lining the renal pyramid also contained large amounts of the isoenzyme while smaller quantities were present in glomeruli and in collecting tubules near the papillary tip. The distribution pattern obtained in tubular epithelia agrees well with that demonstrated in earlier microdissection studies. It is also consistent with the suggestion that glycolysis provides the majority of the energy fuelling the sodium transport mechanisms which form such an essential feature of the countercurrent urine concentration system present within the renal medulla.

Animals↗

PubMed1983

Location in the yeast hexokinase structure of residues related to the enzyme activity.

Seven residues implicated as acting directly in substrate binding in yeast hexokinase B have been identified in the crystallographic structure by chemical sequencing. The cysteine which is regarded as a residue critically maintaining the active conformation of yeast hexokinase has been selectively labelled and likewise located in the structure. In some parts of the amino acid sequence predicted from the high-resolution electron density map it is found that alignments of chemically sequenced peptides can be made unambiguously; however, the extent of matching to the predicted sequence varies considerably along the chain.

Amino Acid Sequence↗

PubMed1990

Low intensity ultrasonic effects on yeast hexokinase.

The kinetics of yeast hexokinase activity exposed to 1 MHz ultrasound of therapeutic intensities 0.1-1.5 W/cm2 was studied using traditional physico-chemical methods and by the thermoinactivation approach. Analysis of the kinetic curves and the kinetic parameters, obtained by two independent methods, suggested specific perturbation processes provoked by the ultrasonic waves, viz., the mechanical breakdown of the contact site between monomer units and the subsequent sonochemical modification of the active enzyme site. Low intensity ultrasound also caused the destabilization of the molecular structure of hexokinase as revealed by the apparent thermolability of the sonicated enzyme.

Hexokinase↗

PubMed1986

Glucose metabolism in fibroblasts from patients with erythrocyte hexokinase deficiency.

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Ki for glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.

Adenosine Triphosphate↗

PubMed1979

Brain microvessel hexokinase: kinetic properties.

Kinetic differences between brain capillary and parenchymal hexokinase in the presence of glucose, ATP. fructose, potassium, sodium and different pH were established. Parenchymal hexokinase is more susceptible to glucose inhibition, can tolerate greater variations in the ATP concentration, is inhibited by increasing concentrations of fructose and potassium, and showed greater activity on the lower pH values. The data suggest that in brain parenchyma and endothelial cells of brain microvessels, there are 2 different enzymes with regard to the kinetics properties.

Adenosine Triphosphate↗

PubMed1996

A novel (TA)n polymorphism in the hexokinase II gene: application to noninsulin-dependent diabetes mellitus in the Pima Indians.

Hexokinase II, one member of a family of structurally similar enzymes that catalyze the phosphorylation of glucose in the 6-position, has been suggested to play a role in the pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM). The gene for hexokinase II, HK2, has been previously mapped to human chromosome 2p13 by fluorescence in situ hybridization, and two-point linkage analysis has placed it near the locus for transforming growth factor alpha, TGFA. We now report the characterization of a (TA)n polymorphism in intron 12 of HK2. Using multipoint analysis of CEPH family genotypes, we have determined the most likely locus order to be cen-D2S169-[D2S286-HK2]-[D2S145-D2S291]-[+ ++D2S45-D2S101-TGFA]-tel. As HKII is a candidate gene that could contribute to the manifestation of insulin resistance and NIDDM, we genotyped 1152 Pima Indians, a Native American tribe that has the highest reported prevalence of NIDDM in the world. Although we did not detect any linkage or association of HK2 with insulin resistance or NIDDM in the Pima Indians, the polymorphism and detailed mapping of HK2 described in this report should prove useful in the assessment of the role of this gene in the predisposition to NIDDM in other populations.

Base Sequence↗

PubMed1985

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme.

Isolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either absorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.

Adenosine Triphosphate↗

PubMed1993

Expression of glucose transporter isoforms (GLUT1, GLUT2) and activities of hexokinase, pyruvate kinase, and malic enzyme in preneoplastic and neoplastic rat renal basophilic cell lesions.

Sequential changes in the expression of two glucose transporter isoforms (GLUT1, GLUT2), and in the activities of hexokinase, pyruvate kinase and malic enzyme during the development of rat renal basophilic cell tumors were studied using histochemical techniques. Early basophilic cell tubules are similar to proximal convoluted tubules (PCT) in their overall histochemical pattern, particularly in the expression of glucose transporters, suggesting that basophilic cell tubules and tumors derived from them arise from PCT. In comparison with PCT, basophilic cell tubules show slightly increased activities of all the enzymes studied. In basophilic cell tumors, markedly elevated hexokinase and pyruvate kinase activities are accompanied by a considerable reduction in the expression of GLUT2. GLUT1 expression is not found in basophilic cell tubules or PCT. Small basophilic cell tumors also do not express GLUT1, but GLUT1 is regularly expressed in several cell layers surrounding necrotic areas within large basophilic cell tumors. Our results indicate that increased glycolytic activity and reduced GLUT2 expression take place during the development of renal basophilic cell tumors.

Animals↗

PubMed1981

A simple procedure to obtain yeast hexokinase free of glucosephosphate isomerase and mannosephosphate isomerase.

A hexokinase preparation was obtained from a Saccharomyces cerevisiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate. The supernatant fraction corresponding to 40-60 % saturation showed the lowest content in GPI and MPI activity. The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose. The results were similar to those obtained with pure commercial hexokinase.

Ammonium Sulfate↗

PubMed1998

Functional consequences of naturally occurring variants of human hexokinase II.

Hexokinase II (HKII) catalyses a key step in glucose metabolism and can be regarded as a candidate gene for insulin resistance and type 2 (non-insulin-dependent) diabetes mellitus. We observed previously four amino acid substitutions among Finnish type 2 diabetic patients: Gln142His, Ala314Val; 0.9%, Arg353Cys; 2.7% and Arg775Gln; 2.7%. The Arg775Gln mutation was also observed in normal control subjects (2.1%) and the Gln142His substitution was found in both Type II diabetic and normal subjects with similar frequencies (approximately 20%). Since Gln at position 142, Ala at 314 and Arg at 775 are present in human and rat hexokinases and could be important for structure and function of the enzyme, we generated all four substitutions by site-directed mutagenesis and expressed them in E.coli. None of these substitutions had any effect on HKII catalytic activity, K(m) or Vmax for glucose values in vitro. Thus unless these substitutions have an impact on enzyme activity or regulation in vivo, it is unlikely that these substitutions contribute to the aetiology of Type II diabetes.

Animals↗

PubMed2005

Involvement of hexokinase Hxk1 in glucose catabolite repression of LIP2 encoding extracellular lipase in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.

Bacterial Proteins↗

PubMed2006

Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism.

Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V(max) with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.

Amino Acid Sequence↗

PubMed1978

An improved assay for hexokinase activity in human tissue homogenates.

An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement, at 340 nm, of NADPH formed according to the reaction scheme: [formula: see text] In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase, EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9; glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.

Glucose 1-Dehydrogenase↗

PubMed1989

Two kinetic methods to study the regulation of mammalian hexokinases.

Two new kinetic analyses for mammalian hexokinases are presented, which permit one to study the regulation of these enzymes by product inhibition. One method uses the pyruvate kinase-coupled assay and the other the glucose-6-phosphate dehydrogenase-coupled assay. Both methods give simple linear plots, which indicate that the magnesium-ATP complex overcomes the glucose 6-phosphate inhibition competitively, but by atypical kinetics. A new regulation coefficient (Kr) was defined and it was shown that, with both assay methods, the reciprocals of the slopes of the simple linear plots are proportional to Kr.[Mg.ATP].NADP, but not NAD, was found to be a powerful inhibitor of pig heart hexokinase.

Animals↗